Antiviral Activities of HIV-1-Specific Human Broadly Neutralizing Antibodies Are Isotype-Dependent.

Broadly neutralizing antibodies (bNAbs) offer promising opportunities for preventing HIV-1 infection. The protection mechanisms of bNAbs involve the Fc domain, as well as their Fab counterpart. Here, different bNAb isotypes including IgG1, IgA1, IgA2, and IgA122 (IgA2 with the hinge of IgA1) were generated and then produced in CHO cells. Their ability to neutralize pseudovirus and primary HIV-1 isolates were measured, as well as their potential ADCC-like activity using a newly developed assay. In our work, gp41-specific IgA seems to be more efficient than IgG1 in inducing ADCC-like activity, but not in its virus neutralization effect. We show that either gp120-specific IgA or IgG1 isotypes are both efficient in neutralizing different viral strains. In contrast, gp120-specific IgG1 was a better ADCC-like inducer than IgA isotypes. These results provide new insights into the neutralization and ADCC-like activity of different bNAbs that might be taken into consideration when searching for new treatments or antibody-based vaccines.

Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.

Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids.

Mycobacterium tuberculosis is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. To better understand the molecular mechanisms by which M. tuberculosis circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of M. tuberculosis of the W/Beijing family to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several M. tuberculosis mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or costimulatory molecule expression by acting as competitive antagonists of Toll-like receptor 2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Our study reveals that producing glycolipid antagonists of pattern recognition receptors is a strategy used by M. tuberculosis to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of M. tuberculosis, considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to M. tuberculosis virulence.

Rational design of adjuvants targeting the C-type lectin Mincle.

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding.

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype-bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Innate immune response of alveolar macrophage to house dust mite allergen is mediated through TLR2/-4 co-activation.

House dust mite, Dermatophagoides pteronyssinus (Der p), is one of the major allergens responsible for allergic asthma. However, the putative receptors involved in the signalization of Der p to the innate immune cells are still poorly defined as well as the impact of their activation on the outcome of the allergen-induced cell response. We previously reported that the HDM activation of mouse alveolar macrophages (AM) involves the TLR4/CD14 cell surface receptor complex. Here using a TLR ligand screening essay, we demonstrate that HDM protein extract engages the TLR2, in addition to the TLR4, in engineered TLR-transfected HEK cells but also in the MH-S mouse alveolar macrophage cell line model. Moreover we found that the concomitant recruitment of the MH-S cell's TLR2 and TLR4 receptors by the HDM extract activates the MyD88-dependent signaling pathway and leads to the secretion of the NF-κB regulated pro-inflammatory factors NO and TNF-α. However unlike with the canonical TLR4 ligand (i.e. the bacterial LPS) mobilization of TLR4 by the HDM extract induces a reduced production of the IL-12 pro-inflammatory cytokine and fails to trigger the expression of the T-bet transcription factor. Finally we demonstrated that HDM extract down-regulates LPS induced IL-12 and T-bet expression through a TLR2 dependent mechanism. Therefore, we propose that the simultaneous engagement of the TLR2 and TLR4 receptors by the HDM extract results in a cross regulated original activation pattern of the AM which may contribute to the Th2 polarization of the allergen-induced immune response. The deciphering of these cross-regulation networks is of prime importance to open the way for original therapeutic strategies taking advantage of these receptors and their associated signaling pathways to treat allergic asthma.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Production and characterization of chimeric anti-HLA monoclonal antibodies targeting public epitopes as tools for standardizations of the anti-HLA antibody detection.

Two chimeric monoclonal antibodies (MoAbs) were designed with variable parts of the mouse antibodies W6/32 and F3.3, and the human constant parts C-gamma1 and C-kappa. These chimeric MoAbs are specific for HLA-class I and HLA-class II public epitopes, respectively. The anti-class I MoAb recognizes all HLA-class I tested so far, and the anti-class II MoAb recognize all HLA-DR, DP, and only DQ antigens of the DQ2 subgroup. These Moabs were used as positive controls in routine tests for the detection of IgG anti-HLA antibodies in the sera of patients (Luminex, flow cytometry, and complement-dependent lymphocytotoxicity assay: CD-LCT). In tests with the LABScreen MIX assay, serial dilutions of the two MoAbs have allowed to determine the thresholds of detection by the Boltzmann sigmoidal regression. The thresholds of detection in mean of fluorescence intensity (MFI) were 926 and 866 for the anti-class I MoAb and the anti-class II MoAb, respectively. The thresholds defined as mean+3SD of MFI values obtained with 30 negative control sera were 411 and 251 for the anti-class I beads and the anti-class II beads, respectively. For the daily validation of the anti-HLA IgG antibodies screening by LABScreen MIX we decided to use the positive control MoAbs at three concentrations (5, 50, and 500ng/ml) and we measured the repeatability (<10%) and reproducibility (<16%) of the method. Used as positive controls in tests with the LABScreen Single Antigen kit, the two MoAbs allowed to estimate daily the quality of beads coated with HLA antigens. The two anti HLA MoAbs were also used as positive control in the cross-match assay by flow cytometry or CD-LCT. In total these two chimeric anti-HLA-MoAbs, which have no equivalent so far, are valuable positive controls for the daily validations of most techniques used for the detection of anti-HLA antibodies according to the good practice guidelines for laboratories.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells.

BACKGROUND: While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. DESIGN AND METHODS: In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. RESULTS: We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. CONCLUSIONS: We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

Long-lasting in vivo gene silencing by electrotransfer of shRNA expressing plasmid.

RNA interference appears as a promising tool for therapeutic gene silencing. A key limit is the delivery of the siRNA. A safe approach is to use a physical method such as in vivo electropulsation with contact electrodes. Getting a long lived silencing can be better approached by using the in situ expression of shRNA. This is presently obtained by using co-electrotransfer of specific plasmids coding for expression and silencing of a fluorescent protein. Using a non invasive fluorescence imaging assay, electrodelivery in mouse muscles is observed to induce complete silencing over more than two months in a specific way. The proper choices of the plasmids (sequence and relative amounts) and of the electric pulsing conditions appear as key parameters in the successful silencing.

Gene therapy based on gemcitabine chemosensitization suppresses pancreatic tumor growth.

Excepting surgical resection, there is no efficient treatment against pancreatic cancer. The chemotherapeutic agent gemcitabine improves the patient's clinical status but survival is not prolonged. The aim of this study was to design a new strategy to render gemcitabine more efficient in the treatment of pancreatic cancer using gene therapy. We have generated a fusion gene (DCK::UMK) combining deoxycytidine kinase (DCK) and uridine monophosphate kinase (UMK), which converts gemcitabine into its toxic phosphorylated metabolite. Antitumor effects of DCK::UMK gene expression were tested in vitro and in vivo in an orthotopic transplantable model of pancreatic cancer established in hamsters. DCK::UMK sensitizes pancreatic cancer cells to gemcitabine by reducing dramatically both in vitro cell viability and in vivo tumor volume. We found that in vivo expression of DCK::UMK resulted in an antitumor bystander effect due to apoptosis of untransduced cells. In vivo intratumoral gene transfer of DCK::UMK using the synthetic carrier PEI induced a potent tumor regression. Taken together, the results show that the fusion gene DCK::UMK sensitizes pancreatic cancer cells to gemcitabine treatment to induce cell death by apoptosis and tumor regression. Intratumoral delivery of the DCK::UMK gene in combination with gemcitabine might be of high interest for pancreatic cancer management.